Bacterial F-type ATP synthases follow a well-choreographed assembly pathway.

F-type ATP synthases are multiprotein complexes composed of two separate coupled motors (F 1 and F O ) generating adenosine triphosphate (ATP) as the universal major energy source in a variety of relevant biological processes in mitochondria, bacteria and chloroplasts. While the structure of many ATPases is solved today, the precise assembly pathway of F 1 F O -ATP synthases is still largely unclear. Here, we probe the assembly of the F 1 complex from Acetobacterium woodii. Using laser induced liquid bead ion desorption (LILBID) mass spectrometry, we study the self-assembly of purified F 1 subunits in different environments under non-denaturing conditions. We report assembly requirements and identify important assembly intermediates in vitro and in cellula. Our data provide evidence that nucleotide binding is crucial for in vitro F 1 assembly, whereas ATP hydrolysis appears to be less critical. We correlate our results with activity measurements and propose a model for the assembly pathway of a functional F 1 complex.